Microbiological demethylation of 7-halo-7-deoxylincomycins

ABSTRACT

A MICROBIOLOGICAL PROCESS FOR THE 1&#39;&#39;-DEMETHYLATION OF 7HALO-7-DEXOYLINCOMYYCINS TO OBTAIN THE CORRESPONDING 7HALO-7-DEOXY-1-DEMETHYLLINCOMYCINS, WHICH ARE KNOWN ACTIVE ANTIBACTERIAL AGENT WITH BROADER SCOPE OF ACTIVITY THAN THE PRESENT COMPOUNDS.

United States Patent O 3,579,426 MICROBIOLOGICAL DEMETHYLATION F 7-HALO-7-DEOXYLINCOMY'CINS Alexander D. Argoudelis, John H. Coats, Donald J.

Mason, and Oldrich K. Sebek, Kalamazoo, Micl 1., assignors to The Upjohn Company, Kalamazoo, Mich. No Drawing. Filed Oct. 21, 1968, Ser. No. 769,411 Int. Cl. C12d 9/00 U.S. Cl. 195-80 7 Claims ABSTRACT OF THE DISCLOSURE A microbiological process for the 1'-demethylation of 7 halo-7-deoxylincomycins to obtain the corresponding 7- halo-7-deoxy-l-demethyllincomycins, which are known active antibactreial agents with broader scope of activity than the present compounds.

BACKGROUND OF THE INVENTION This invention provides an effective process for the production of 7-halo-7-deoxy 1 demethyllincomycms, which were heretofore available only by a long and com plicated chemical methods of synthesis.

SUMMARY OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION The microbiological process of this invention is represented by the following reaction scheme:

H i N 3 1 iI|-NH 0 R1 0 wherein R is methyl or ethyl, and R is an alkyl of from 2 to 8 carbon atoms, inclusive, and X is chlorine or bromine.

Examples of alkyl of from 1 to 8 carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl and isomeric forms thereof.

In this application, the wavy line (I) appearing at the 4'- position of the pyrrolidine ring is used to indicate that the group R can .be in position cis (below the plane of the ring or trans (above the plane of the ring), with respect to the carbonyl group and the wavy line appearing at the 7-position is used to indicate that both halo epimers are to be included, i.e,, the (S) configuration wherein the halo group is located to the right as shown in the formulae or the (R) configuration wherein the halo group is located to the left.

'ice

The compounds of Formulas I and H exist either in the protonated or non-protonated forms. The free bases can be converted to stable acid-addition salts in accordance with methods known in the art, for example, by neutralizing the free base with the appropriate acid to below about pH 7.0, and advantageously to about pH 2 to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, 3-phenylsalicyclic, 5-phenylsalicyclic, 3-methylglutaric, orthosulfobenzoic, cyclopentanepropionic, 1,2-cyclohexanedicarboxylic, 4 cyclohexanecarboxylic, otadecenylsuccinic, octenylsuccinic, methanesulfonic, benzenesulfonic, helianthic, Reineckes dimethyldithiocarbarnic, cyclohexylsulfamic, hexadecylsulfamic, octadecylsulfamic, sorbic, monochloroacetic, undecylenic, 4-hydroxyazobenzene-4-sulfonic, octyldecylsulfuric, picric, benzoic, cinnamic, and like acids.

The 1'-demethyl compounds produced by the process of the present invention, represented by Formula II, above, and the acid addition salts thereof are known active antibacterial agents having improved gram negative activity over the corresponding compounds of Formula I. In addition 7(S)-chl0r0-7-de0xy-1'-demethyllincomycin and related compounds are active antimalarial agents.

The starting compounds of Formula I, above, and the acid addition salts thereof, are known in the art, see for example, U.S. Pat. 3,380,992, Belgium Pats. 676,202 and 705,345 issued Aug. 8, 1966, and Apr. 19, 1968, respectively; French Pat. 1,474,237, issued Feb. 13, 1967; South African Pats. 66/319 and 66/320, issued Jan. 25, 1967, and Magerlein et al., Chemical Modifications of Lincomycin, Antibiotic and Chemotherapy (1966), page 727- 736.

The microbiological process of this invention comprises subjecting a 7-halo-7-deoxylincomycin (I) to the activity of one of the following microorganisms:

Streptomyces punipalus sp. nov., NRRL 3529; Streptomyces venezuelae, NRRL 3528; Streptomyces venezuelae, NRRL 3527; Aspergillus ornatus, NRRL 2291; Scopulariopsis brevicaulis, ATCC 7903;

T richoderma viride, NRRL 1762; Streptomyces spectabilis, NRRL 2494; and Streptomyces speclabilis, NRRL 2792 Streptomyces punipalus sp. nov. NRRL 3529.

The materials and methods used in characterizing this culture were those cited in the following references:

Dietz, A., Streptomyces stefiisburgensis sp. nov., J. Bacteriol. 94: 2022-2026, 1967.

Shirling, E. B., and D. Gottlieb, Methods for characterization of Streptomyces species, Intern J. System. Bacteriol. 16: 313-340, 1966.

DESCRIPTION Color characteristics.Pink to pink tan aerial mycelium. Melanin-positive. Appearance on Ektachrome is given in Table 1. Reference color characteristics on agar media are given in Table 2. The culture may be placed in the Red (R) and Yellow (Y) color series of Tresner and Backus (Appl. Microbiol. 11: 335-338, 1963).

Microscopic characteristics-Sporophores are open spiral (RA) to spiral (S) in the sense of Pridham et al. (Appl. Microbiol. 6: 52-79, 1958). Sporophores are usually tightly coiled at the tip. Spores are more than 50 in a chain, smooth and sometimes phalangiform (J. Bacteriol. 91: 1998-2005, 1966). The spore surface is ridged and has much surface detail.

Cultural and biochemical characteristics.-See Table 3.

Carbon utilization.The ability of the culture to grow on carbon compounds was determined in the synthetic medium of Pridham and Gottlieb (J. Bacteriol. 56: 107- 114, 1948) and in their medium as modified by Shirling and Gottlieb (Intern. 1. System Bacteriol. 16: 313-340, 1966). In the former growth was good on D-Xylose, L- arabinose, D-fructose, D-galactose, D-glucose, D-mannose, maltose, cellobiose, dextrin, soluble starch, glycerol, inositol, sodium acetate, sodium citrate, and sodium succinate; moderate on the control, rhamnose, sucrose, lactose, rafiinose, inulin, dulcitol, D-mannitol, D-sorbitol, and salicin; slight on phenol, cresol, sodium formate, sodium tartrate, and solium salicylate. There was no growth on sodium oxalate. In the latter, there was no growth on the plain control and good growth on the glucose control. Growth was better than on the glucose control on L-arabinose, somewhat less than with glucose on D-xylose and inositol, and negative on D-mannitol, D- fructose, rhamnose, raflinose, and cellulose.

Temperature.-Good growth with sporulation occurs at 18-37 C. on Bennetts, Czapeks sucrose, and maltosetryptone agars.

Streptomyces punipalus sp. nov., NRRL 3529, is an actinomycete of the genus Streptomyces which was isolated from a soil sample. The culture was differentiated from known Stretptomyces species. This soil isolate has a distinctive pink color pattern not seen in any validly described, publicly available Streptomyces, sporophores that are readily distinguished from those of other pink sporulating, melanin-positive streptomycetes, and an ability to grow and sporulate well at temperatures of 18-37 C. The distinctive properties of the culture require it to be considered a new species of Streptomyces designated S treplomyces punipalus sp. nov., NRRL 3529. The culture has been named to conform with the International Code of Nomenclature of Bacteria (Intern. J. Bacteriol. 16: 459-490, 1966). This organism has been deposited, and is available without restriction as NRRL 3529, from the Northern Utilization Research and Development Branch, US. Department of Agriculture, Peoria, 111.

TABLE 1 [Appearance of Streptomyccs punipalus on Ektachrome] TABLE 2 [Reference color characteristics of Streptomyces punipaias] E. Jacobson, et a]. Color Harmony Manual 3d ed, 1948, Container Corp. of America Agar medium K. L. Kelly et a1. 1800- NB S method of designating colors and a dictionary of color names, 1955, U.S. Dept. of Commerce Cir. 5153,1955

Inorganic-salts starch (TSP-4) Glycerol-asparagine (I SP-5) 5cb(g} (no name) 2gc bamboo, chamois 2ec(g) biscuit, ecru, oatmeal, sand"... 3cb(g) sand 2ec(g) biscuit, ecru, oatmeal, sand 2ge(g) covert tan, griege S 3ba(g) pearl, shell tint 2gc(g) bamboo, chamois.

gm grayish yellow.

Do. Do.

94 in light olive brown. 109 light grayish yellow.

90 gm grayish yellow. 94 in light olive brown. 109 gm light grayish yellow.

5cb(g) (no name) 2gc(g) bamboo, charnois 90 gm grayish yellow.

2ge(g) covert tan, griege 9 2 in light olive brown.

109 gm light grayish yellow.

5cb(g) (no name) 3ca(g) pearl pink, shclL.

R 2ca(g) light ivory eggshell P 2ec(g) biscuit, ecru, oatmeal, sand S 4ca(g) flesh pink pearl pink, shell pink, tea- 28 g light yellowish pink.

rose- R 2ca(g) light ivory eggshell P 2eetg) biscuit, ccru, oatmeal, sand 73 gm pale orange yellow.

28 g light yellowish pink.

31 gm pale yellowish pink. 89 gm pale yellow.

90 gm grayish yellow.

31 gm pale yellowish pink. 89 gm pale yellow. 90 gm grayish yellow.

Notc.-S=Surlaee; P=Pigment; R= Reverse; (g)=glossy surface (otherwise matte).

TABLE 3 [Cultural characteristics of Streptomyces pum'palus] Medium Surface Reverse Other Agar media:

Peptone-iron Gray Brown Melanin-positive.

Calcium-malate Trace gray-.ink Pale cream No pgment; Malate solbilize Glucose-asparagine White with trace gray Yellow No pigment.

Skim milk Gray-white on periphery Olive-tan Olive-tan pigment; casein not solubilized.

Tyrosine Gray-pink Tan Tan pigment; tyrosine solubilized.

Xanthine do Yellow Pale yellow-tan pigment;

Xanthine solubilized around growth.

Nutrient starch do d0 Pale olive-yellow pigment;

starch not hydrolyzed.

Yeast extract-malt extract do Tan Pale tan pigment.

Bennetts U Pink-tan" Maltese-tryptone. Pale pink-tan,

Peptone-yeast extraetnou N0 surface growth USP-6). Tyrosine (TSP-7) Rose-pink Pink-tan No pigment.

TABLE 3-Cbnftinued Medium Surface Reverse Other Gelatin media:

Plain Pale pink aerial growth on Tan pigment M; olive surface ring. pigment liquefaction complete.

Nutrient "do Tan pigment M; olive pigment liquefaction complete.

Broth media:

Nutrient nitrate Trace pink-White aerial 0n Yellow-tan pigment; floccuurface ring. lent growth at base;

nitrate reduced to nitrite.

Synthetic nitrate Compact growth at base;

nitrate reduced to nitrite.

Litmus milk Gray-pink aerial growth on No peptonization; no reheavy gray-green duction; color blue-gray; surface nng. pH 7.

The operational conditions and reaction procedures for the bioconversion process of this invention are advantageusly those known in the art of bioconversion as illustrated in Murray et al., US. Pats. 2,602,769 and 2,735,800.

In the practice of this invention, the bioconversion can be effected by a growing or resting culture of the microorganism or by sporm, Washed cells or enzymes of the microorganism.

Culture of the microorganism for the purpose and practice of this invention is in or on a medium favorable to its development. Sources of nitrogen and carbon should be present in the culture medium and an adequate sterile air supply should be maintained during the conversion, for example, by the conventional techniques of exposing a large surface of the medium or by passing air through a. submerged culture.

Nitrogen in assimilable form can be provided by sources normally employed in such processes, such as corn steep liquor, cotton seed meal, soybean metal, yeast extracts, peptone, soluble or insoluble vegetable or animal protein, lactalbumin, casein, whey, distillers solubles, amino acids, nitrates and ammonium compounds, such as ammonium tartrate, nitrate, sulfate and the like.

Available carbon can also be provided by sources normally used in bioconversions such as carbohydrates, e.g., glucose, fructose, sucrose, lactose, maltose, dextrines, starches; meat extracts, peptones, amino acids, proteins, fatty acids, glycerol, Whey and the like. These materials may be used either in a purified state of as concentrates such as whey concentrate, corn steep liquor, grain mashes, cotton seed meal, and the like, or as mixtures of the above. Many of the above sources of carbon can also serve as a source of nitrogen.

The medium can desirably have a pH before inoculation of between about pH 4 to about 8 though a higher or lower pH can be used. A temperature between about 25 to 32 C. is preferred for growth of the microorganism but higher or lower temperatures within a relatively wide range are suitable.

The substrate compound (I) can be added to the culture during the growth period of the microorganism as a single feed or by intermittent addition during the conversion period, or it can be added to the medium before or after sterilization or inoculation making appropriate adjustments for effects of pH and/or temperature upon the stability of the substrate used. The preferred, but not limiting, range of concentration of the substrate in the culture medium is about 50500 mg. per liter. In the practice of this invention, it is convenient to add the substrate to the medium in the form of a water soluble acid addition salt.

The temperature during the fermentation can be the same as that found suitable for growth of the microorganism. It need be maintained only within such range as supports life, active growth or the enzyme activity of the microorganism. A range of to 35 C. is preferred. A pH of about 6 to 8 is generally preferred for growth of the microorganism during the bioconversion. Aeration can be effected by surface culture or preferably by use of submerged fermentation conditions with air sparging,

in accordance with methods well known in the art. The time required for demethylation by the enzymatic system of the microorganism employed can vary considerably. The range of about 2 to hours is practical but not limiting; 24-72 hours is generally satisfactory. The progress of the bioconversion and its completion are conveniently determined by paper-strip chromatography, or thin-film chromatography [Heftman, Chromatography (1961), Reinhold Publishing Co., New York, N.Y.].

Alternatively, demethylation of the selected substrate can be effected by subjecting it to the activity of enzymes prepared from the microorganism, to the action of spores of the microorganism, and to the action of isolated cells of the microorganism. Isolated enzyme preparations can be prepared in accordance with the general procedure disclosed by Zuidweg et al., Biochim, Biopy. Acta, 58, 131-133 (1962). The bioconversion can be effected with spores in accordance with the general process disclosed in US. Pats. 3,031,379 and 3,031,382. The separation of washed cells from the fermentation medium is well known in the art, see for example, US. Pat. 2,831,789.

The term demethylation as used throughout this specification means the enzymatic action of a growing or resting culture of the microorganism which effects removal of a methyl group from the molecule of the substrate under fermentation conditions.

After completion of the fermentation, the resulting product (H) is recovered from the fermentation beer by conventional methods. For example, the whole beer can be extracted with a Water-immiscible organic solvent such as methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, ether, amyl acetate, benzene, and the like, or the beer and mycelium can be separated by conventional methods such as centrifugation or filtration, and then separately extracted with suitable solvents. The mycelium can be extracted with either watermiscible or water-immiscible solvents or in cases where little or no product is contained in the mycelium, it can be merely washed with water and the wash water added to the beer filtrate. The beer, free of mycelium, can then be extracted with water-immiscible solvents such as those listed above. The extracts are combined, dried over a drying agent such as anhydrous sodium sulfate, and the solvent removed by conventional methods such as evaporation or distillation at atmospheric or reduced pressure.

Alternatively, the product can be adsorbed from the beer on an adsorbent non-ionic resin or on carbon and the product eluted with a polar organic solvent such as methanol, ethanol, acetone, ethyl acetate, methyl ethyl ketone, aqueous mixtures thereof, and the like.

The 1'-demethyl products (II) obtained by either the extraction or elution procedures can be isolated and further purified by conventional methods, e.g., chromatography and/or crystallization, and the like. The products (II) can be obtained as free bases or as acid addition salts in accordance with procedures hereintofore disclosed.

The following examples are intended to illustrate the process of this invention. The examples are for the purpose of illustrating the best mode contemplated of carrying out the invention and to supplement the foregoing disclosure with additional descriptions of the manner and process of carrying out the invention so as to further enable workers skilled in the art to do so, but they are not to be construed as limiting.

EXAMPLE 1 7 (S) -chlr0-7-deoxy-l '-de methyllincomycin A vegetative seed from a culture of Streptomyces punipalus, NRRL 3529; is prepared by growing the culture for 3 days at about 28 C. in a reciprocating shake flasks (250 r.p.m., stroke 2 /2 cm.) on a medium consisting of 25 g. per liter of Cerelose (glucose monohydrate) and 25 g. per liter of Pharmamedia (cotton seed meal), Traders Protein Oil Mill Co., P.O. Box 1837, Fort Worth, Tex. 76100 in tap water. The medium is sterilized prior to inoculation.

The resulting seed is used to inoculate a medium consisting of 20 g. per liter of black strap molasses, 30 g. per liter of dextrin, 15 g. per liter of fish meal and 15 g. per liter of Pharmamedia. The medium is adjusted to pH 7.2 and 120 shake flasks (500 ml.) are prepared each containing 100 ml. of the medium. The shake flasks are sterilized (pH'after sterilization is 6.2) and are inoculated with 5 ml. of the vegetative seed (prepared above) per 100 ml. of medium.

The culture is then allowed to grow for 48 hours on a rotary shaker (250 rpm. stroke 2.5 cm.) at about 28 C. At the end of the 48-hour growth period, 5 mg. of 7(8)- chloro-7-deoxylincomycin hydrochloride is added to each shake flask and the fermentation is continued for an additional period of about 24 hours. The contents of the flasks are then pooled (about 10 liters), diatomaceous earth is added and the beer and mycelium are separated by filtration. The filter cake is washed with l l. of water and the wash water is added to the clear beer.

The clear beer thus obtained is passed over a column containing Amberlite XAD-2 resin (Rohm and Haas Co., Philadelphia, Penn. 19105); the column is prepared by wet packing with 350 g. of the resin and allowing the resin to setle by gravity. The bed-volume of the column is 500 ml. The clear beer is added at the top of the column and passed through the resin at a flow rate of about 4% of the bed volume. The column is then eluted with 3.5 liters of methylethyl ketone: water (95:5 v./v.). Fractions of ml. each are collected and aliquots spotted on agar trays seeded with Sarcina lutea. Those fractions which inhibit the growth of S. lurea, are combined and the upper and lower phases are separated. The upper phase is concentrated to dryness and labeled preparation A, the lower aqueous phase is adjusted to pH 10 with 20 g. of sodium carbonate per 100 ml. of the lower phase. The alkaline solution thus obtained is then extracted with methylene chloride and the methylene chloride solution is evaporated to dryness and labeled preparation B. Thin layer chromatography [silica gel G, methylethyl ketone: acetone: water (l40:40:22 v./v.)] shows the presence of 7(S) chloro 7 deoxylincomycin and 7(8) chloro- 7-deoXy-l'-demethyllincomycin in both preparations A and B.

Preparations A and B (6 g.) are combined, dissolved in about 10 ml. of chloroformzmethanol (6:1 v./v), mixed with 24 g. of silica gel (Merck-Darmstadt No. 7734), and evaporated to dryness. The dry material thus obtained is then added at the top of a wet packed (chloroform-methanol 6:1 v./v.) column containing 600 g. of the same silica gel. Additional silica gel is added at the top of the load bed and the column is eluted with about 12.8 I. of chloroform-methanol (6:1 v./v.). Fractions of 20 ml. are collected and tested for bioactivity on agar trays seeded with S. lutea. The. fractions which are shown by thin layer chromatography to contain 7(S)-chloro-7- deoxy l' demethyllincomycin were combined and the 7 (S) chloro 7 deoxy 1 demethyllincomycin is further identified by comparison with an authentic sample of the same compound using thin layer and paper 8 chromatography and by comparison of the mass spectrum of the bioconversion product with that of the authentic sample of 7(5)-chloro-7-deoxy-l-demethyllincomycin.

In the same manner, the following microorganisms:

Streptomyces venezuelae, NRRL 3528; Streptomyces venezuelae, NRRL 3527; Streptomyces spectabilis, NRRL 2494; Streptomyces spectabilis, NRRL 2792; Aspergillus ornams, NRRL 2291; Scopulariopsis brevicaulis, ATCC 7903; and Trichoderma viride, NRRL 1762;

are used in place of Streptomyces punipalus, NRRL 3529; to obtain 7(5) chloro 7 deoxy 1' demethyllincomycin or its acid addition salts.

In the same manner following the procedure of Example 1, other 7 chloro 7 deoxylincomycins, of Formula I are substituted as the substrate in place of 7(8)- chloro 7 deoxylincomycin to obatin the corresponding 7 chloro 7 deoxy 1' demethyllincomycins of Formula II. The following conversions are representative of further substrate materials which are used, either as free bases or acid addition salts with reference to Formula I, in this invention to obtain the corresponding l-demethyl compounds of Formula II:

R1 7-X R Trans-ethyl (S)-Cl Methyl. Do. I (R)Cl Do. Cls-ethyl I (S)-Br Do. Do, (R)-Ol Do. Trans-n pro (S)-C Do.

Do. (S)Br Ethyl. Do (R)-Cl Methyl. Cis-n-propyl (S)Cl D0. Trans-i-propyl (8)431 Do. Trans-nbut-yl (S)Cl D0.

0 (S)Br Ethyl. Do (R)Br Methyl. Cisu-butyl. (S)-Cl Do. Trans-n-pentyl (S)-Gl Do.

D (S)Cl Ethyl.

(R)-Cl Methyl. Cis-n-pentyl. Do.

Trans-n-heXyL De Trans-i-oetyl. Cis-n-oc-tyl (S EXAMPLE 2 4-despropyl-4'-trans-n-pentyl-7-deoxy-7 (S) chloro-1 -demethyllinc0mycin The bioconversion and recovery procedures of Example 1 are repeated by using the same microorganism but substituting 4 despropyl 4 trans n pentyl 7 deoxy 7( S) chlorolincomycin hydrochloride as the substrate to obtain 4' despropyl 4' trans n pentyl- 7-deoXy-7 (S) -chlorol'-dernethyllincomycin.

EXAMPLE 3 7 (S) -chl0r0-7-de0xy-1 '-demethyllincomycin After 2 to days when a heavy growth of mycelium is apparent by visual observations, 50-100 mg. of 7(S) chloro-7-deoxylincomycin hydrochloride are added to each flask and the incubation of each flask is continued for an additional period of from 3 to 5 days.

The samples are then checked for bioconversion using ascending paper chromatography. Aliquots of 2 to 5 ,ul. of the filtered beer from each of the bioconversions is applied on filter paper (Whatman No. 3 HR, 18 x 13 mm.). The bioconversion products are separated from the starting material by ascending chromatography using a mixture of ethyl acetate; acetone; water (8:5 :1 v./v.) as solvent system. Comparison of these papergrams with a papergram made in the same manner from an authentic mixture of 7(S)-chloro-7-deoxylincomycin and 7(5)- chloro-7-deoxy-1-demethyllincomycin shows that each of the bioconversions produce 7(S)-chloro-7-deoxy-l'-demethyllincomycin as the bioconversion product.

Likewise, using the microorganisms listed above, other analogs of 7(S)-chloro-7-deoxylincomycin and 7 (R)- chloro-7-deoxylincomycin, represented by Formula I, above, for example, those compounds listed in Example 1, above are converted to the corresponding 1-demethyl compounds of Formula II.

EXAMPLE 4 7 (S) -chloro-7-de oxy-l -d emethyllincomycin Two ml. portions of a medium having the same composition as that described in Example 1 above, are placed in l ml. flasks and sterilized. One is inoculated with Streptomyces venezuelae, NRRL 3528; and the other with Streptomyces venezuelae, NRRL 3527. After about 3 days when a heavy growth of mycelium is apparent, 1 mg. of 7(S)-chloro-7-deoxylincomycin hydrochloride are added to each flask and the fermentation is continued for an additional period of about 3 days.

The samples are then checked for bioconversion by paper chromatography by the method described in Example 3, above. The bioconversion products in each are identified as 7 (S)-chloro-7-deoxy-1-demthylincomycin.

We claim:

1. The process for the production of a l'-demethyl compound of the formula:

wherein R is methyl or ethyl, R is alkyl of from 2 to 8 carbon atoms, inclusive, and X is chlorine or bromine,

which comprises subjecting a 1-methy1 compound of the formula:

wherein R, R and X have the meanings given above, or an acid addition salt thereof, to the demethylating activity of Streptomyces punipalus, NRRL 3529; Streptomyces venezuelae, NRRL 3528; Streptomyces venezuelae, NRRL 3527; Streptomyces spectabilis, NRRL 2494; Streptomyces spectabilis, NRRL 2792; Aspergillus ornatus, NRRL 2291; Scapulariopsis brevicaulis, ATCC 7903; or Trichoderma viride, NRRL 1762.

2. The process of claim 1, wherein the demethylation is carried out under submerged fermentation conditions and the fermentation is continued until a substantial amount of demethylated product is produced.

3. The process of claim 1, wherein the demethylation is carried out under submerged fermentation conditions; the fermentation is continued until a substantial amount of demethylated product is produced and the demethylated product is recovered from the fermentation medium.

4. The process of claim 1, wherein the demethylation is carried out in an aqueous nutrient medium under aerobic fermentation conditions.

5. The process of claim 1, wherein the demethylation is carried out in an aqueous nutrient medium under submerged fermentation conditions with Streptomyces punipalus, NRRL 3529.

6. The process of claim 1, wherein 7(S)-chloro-7-deoxylincomycin hydrochloride is subjected to the demethylating activity of Streptomyces punipalus, NRRL 3529, in an aqueous nutrient medium under submerged fermentation conditions to obtain 7(S)-chloro-7-deoxy-1-demethylincomycin.

7. The process of claim 1, wherein 4-despropyl-4'- trans-n-pentyl-7-deoxy-7 (S -chlorolincomycin hydrochloride is subjected to the demethylating activity of Streptomyces punipalus, NRRL 3529, in an aqueous nutrient medium under submerged fermentation conditions to obtain 4'-despropyl-4-trans-n-pentyl-7-deoxy-7 (S)-chlorolincomycin.

References Cited UNITED STATES PATENTS 3,380,992 4/1968 Argoudelis et al -80X 3,520,778 7/1970 Bellet 195-80X JOSEPH M. GOLIAN, Primary Examiner U.S.Cl.X.R. 

